Epidermal Growth Factor Receptor Overexpressing Cells Research Articles

Multivalent Ligand-Nanoparticle Conjugates Amplify Reactive Oxygen Species Second Messenger Generation and Enhance Epidermal Growth Factor Receptor Phosphorylation.

The epidermal growth factor (EGF) receptor (EGFR) is heterogeneously distributed on the cellular surface and enriched in clusters with diameters of tens of nanometers. Multivalent presentation of EGF ligand on nanoparticles (NPs) provides an approach for controlling and amplifying the local activation of EGFR in these clusters. Reactive oxygen species (ROS) have been indicated to play a role in the regulation of EGFR activation as second messengers, but the effect of nanoconjugation on EGF-mediated ROS formation and ROS-induced EGFR activation is not well established. The goal of this manuscript is to characterize the multivalent enhancement of EGF-induced ROS formation and to test its effect on EGFR phosphorylation in breast cancer cell models using gold (Au) NPs with a diameter of 81 ± 1 nm functionalized with two different EGF ligand densities (12 ± 7 EGF/NP (NP-EGF12) and 87 ± 6 EGF/NP (NP-EGF87)). In the EGFR overexpressing cell lines MDA-MB-231 and MDA-MB-468, NP-EGF87 achieved a measurable multivalent enhancement of ROS that peaked at concentrations c ROSmax ≤ 25 pM and that were EGFR and nicotinamide adenine dinucleotide phosphate oxidase (NOX) dependent. NP-EGF12 failed to generate comparable ROS levels as NP-EGF87 in the investigated NP input concentration range (0-100 pM). In cells with nearly identical numbers of bound NP-EGF87 and NP-EGF12, the ROS levels for NP-EGF87 were systematically higher, indicating that the multivalent enhancement is exclusively related not only to avidity but also to a stronger stimulation per NP. Importantly, the increase in EGF-induced ROS formation associated with EGF nanoconjugation at c ROSmax resulted in a measurable gain in EGFR phosphorylation, confirming that ROS generation contributes to the multivalent enhancement of EGFR activation in response to NP-EGF87.

Photosensitive EGFR-Targeted Nanocarriers for Combined Photodynamic and Local Chemotherapy.

The major limitation of any cancer therapy lies in the difficulty of precisely controlling the localization of the drug in the tumor cells. To improve this drawback, our study explores the use of actively-targeted chemo-photo-nanocarriers that recognize and bind to epidermal growth factor receptor-overexpressing cells and promote the local on-demand release of the chemotherapeutic agent doxorubicin triggered by light. Our results show that the attachment of high concentrations of doxorubicin to cetuximab-IRDye700DX-mesoporous silica nanoparticles yields efficient and selective photokilling of EGFR-expressing cells mainly through singlet oxygen-induced release of the doxorubicin from the nanocarrier and without any dark toxicity. Therefore, this novel triply functionalized nanosystem is an effective and safe nanodevice for light-triggered on-demand doxorubicin release.

Ru(II) complexes as potent photosensitizers in Photodynamic Therapy

s / Photodiagnosis and Photodynamic Therapy 12 (2015) 325–375 339 for photodynamic therapy in cancer and a potential alternative treatment to conventional chemotherapeutics, such as cisplatin. http://dx.doi.org/10.1016/j.pdpdt.2015.07.057 Synthesis of a new bacteriochlorin derivative with long-wavelength absorption: A potential dye for use as a photosensitizer Francisco F. de Assis, Timothy J. Brocksom, Kleber T. de Oliveira Department of Chemistry, Federal University of Sao Carlos, Brazil Bacteriochlorins are very attractive candidates for use in photodynamic therapy (PDT) due to their important photophysical properties and absorption bands at the infrared region. These compounds canbeobtained fromnatural sources, suchas cyanobacteria and are denominated bacteriochlorophylls. However, the syntheses of these structures are very laborious and represent a challenge from the synthetic point of view. In our research, we have prepared a new bacteriochlorin compound, which presents a strong absorption band around 800nm. This structure was obtained by the Sonogashira cross-coupling between a tetrabromo bacteriochlorin, prepared by Lindsey’smethodology and phenylacetylene. Currently, we are evaluating the photophysical properties of this new compound as a photosensitizer for use in PDT treatment. http://dx.doi.org/10.1016/j.pdpdt.2015.07.058 Nanobody-targeted photodynamic therapy for oncology Raimond Heukers, Paul M.P. van Bergen en Henegouwen, Sabrina Oliveira Molecular Oncology, Division of Cell Biology, Department of Biology, Faculty of Science, Utrecht University, The Netherlands Monoclonal antibodies have been employed to target photosensitizers (PS) and improve tumor selectivity in photodynamic therapy (PDT). However, antibody-PS conjugates have a long blood half-life and a relatively poor tumor penetration, which result in long photosensitivity in patients and limited therapeutic efficacy. In an attempt to target PS specifically and homogenously to tumors and to accelerate PS clearance, we have developed new conjugates consisting of nanobodies targeting the epidermal growth factor receptor (EGFR) and a traceable PS (IRDye700DX). Nanobodies are the variable domain of heavy-chain antibodies existent in camelids and have a great potential in cancer imaging and therapy. The selected PS is a silicon-phthalocyanine derivative that is more hydrophilic than common PS and is suitable for nearinfrared imaging. The fluorescent nanobody-PS conjugates allow the distinction of cell lineswith different expression levels of EGFR. These conjugates specifically induce cell death of EGFR overexpressing cells, while PS alone or the nanobody-PS conjugates in absence of light induce no toxicity. Delivery of PS using internalizing biparatopic nanobody-PS conjugates results in even more pronounced toxicities. Initial in vivo studies point towards a potent therapy, enabling the combination with molecular imaging, which can have a significant impact in the field of targeted PDT. http://dx.doi.org/10.1016/j.pdpdt.2015.07.059 Ru(II) complexes as potent photosensitizers in Photodynamic Therapy

Nanobody-targeted photodynamic therapy for oncology

s / Photodiagnosis and Photodynamic Therapy 12 (2015) 325–375 339 for photodynamic therapy in cancer and a potential alternative treatment to conventional chemotherapeutics, such as cisplatin. http://dx.doi.org/10.1016/j.pdpdt.2015.07.057 Synthesis of a new bacteriochlorin derivative with long-wavelength absorption: A potential dye for use as a photosensitizer Francisco F. de Assis, Timothy J. Brocksom, Kleber T. de Oliveira Department of Chemistry, Federal University of Sao Carlos, Brazil Bacteriochlorins are very attractive candidates for use in photodynamic therapy (PDT) due to their important photophysical properties and absorption bands at the infrared region. These compounds canbeobtained fromnatural sources, suchas cyanobacteria and are denominated bacteriochlorophylls. However, the syntheses of these structures are very laborious and represent a challenge from the synthetic point of view. In our research, we have prepared a new bacteriochlorin compound, which presents a strong absorption band around 800nm. This structure was obtained by the Sonogashira cross-coupling between a tetrabromo bacteriochlorin, prepared by Lindsey’smethodology and phenylacetylene. Currently, we are evaluating the photophysical properties of this new compound as a photosensitizer for use in PDT treatment. http://dx.doi.org/10.1016/j.pdpdt.2015.07.058 Nanobody-targeted photodynamic therapy for oncology Raimond Heukers, Paul M.P. van Bergen en Henegouwen, Sabrina Oliveira Molecular Oncology, Division of Cell Biology, Department of Biology, Faculty of Science, Utrecht University, The Netherlands Monoclonal antibodies have been employed to target photosensitizers (PS) and improve tumor selectivity in photodynamic therapy (PDT). However, antibody-PS conjugates have a long blood half-life and a relatively poor tumor penetration, which result in long photosensitivity in patients and limited therapeutic efficacy. In an attempt to target PS specifically and homogenously to tumors and to accelerate PS clearance, we have developed new conjugates consisting of nanobodies targeting the epidermal growth factor receptor (EGFR) and a traceable PS (IRDye700DX). Nanobodies are the variable domain of heavy-chain antibodies existent in camelids and have a great potential in cancer imaging and therapy. The selected PS is a silicon-phthalocyanine derivative that is more hydrophilic than common PS and is suitable for nearinfrared imaging. The fluorescent nanobody-PS conjugates allow the distinction of cell lineswith different expression levels of EGFR. These conjugates specifically induce cell death of EGFR overexpressing cells, while PS alone or the nanobody-PS conjugates in absence of light induce no toxicity. Delivery of PS using internalizing biparatopic nanobody-PS conjugates results in even more pronounced toxicities. Initial in vivo studies point towards a potent therapy, enabling the combination with molecular imaging, which can have a significant impact in the field of targeted PDT. http://dx.doi.org/10.1016/j.pdpdt.2015.07.059 Ru(II) complexes as potent photosensitizers in Photodynamic Therapy

Synthesis of a new bacteriochlorin derivative with long-wavelength absorption: A potential dye for use as a photosensitizer

s / Photodiagnosis and Photodynamic Therapy 12 (2015) 325–375 339 for photodynamic therapy in cancer and a potential alternative treatment to conventional chemotherapeutics, such as cisplatin. http://dx.doi.org/10.1016/j.pdpdt.2015.07.057 Synthesis of a new bacteriochlorin derivative with long-wavelength absorption: A potential dye for use as a photosensitizer Francisco F. de Assis, Timothy J. Brocksom, Kleber T. de Oliveira Department of Chemistry, Federal University of Sao Carlos, Brazil Bacteriochlorins are very attractive candidates for use in photodynamic therapy (PDT) due to their important photophysical properties and absorption bands at the infrared region. These compounds canbeobtained fromnatural sources, suchas cyanobacteria and are denominated bacteriochlorophylls. However, the syntheses of these structures are very laborious and represent a challenge from the synthetic point of view. In our research, we have prepared a new bacteriochlorin compound, which presents a strong absorption band around 800nm. This structure was obtained by the Sonogashira cross-coupling between a tetrabromo bacteriochlorin, prepared by Lindsey’smethodology and phenylacetylene. Currently, we are evaluating the photophysical properties of this new compound as a photosensitizer for use in PDT treatment. http://dx.doi.org/10.1016/j.pdpdt.2015.07.058 Nanobody-targeted photodynamic therapy for oncology Raimond Heukers, Paul M.P. van Bergen en Henegouwen, Sabrina Oliveira Molecular Oncology, Division of Cell Biology, Department of Biology, Faculty of Science, Utrecht University, The Netherlands Monoclonal antibodies have been employed to target photosensitizers (PS) and improve tumor selectivity in photodynamic therapy (PDT). However, antibody-PS conjugates have a long blood half-life and a relatively poor tumor penetration, which result in long photosensitivity in patients and limited therapeutic efficacy. In an attempt to target PS specifically and homogenously to tumors and to accelerate PS clearance, we have developed new conjugates consisting of nanobodies targeting the epidermal growth factor receptor (EGFR) and a traceable PS (IRDye700DX). Nanobodies are the variable domain of heavy-chain antibodies existent in camelids and have a great potential in cancer imaging and therapy. The selected PS is a silicon-phthalocyanine derivative that is more hydrophilic than common PS and is suitable for nearinfrared imaging. The fluorescent nanobody-PS conjugates allow the distinction of cell lineswith different expression levels of EGFR. These conjugates specifically induce cell death of EGFR overexpressing cells, while PS alone or the nanobody-PS conjugates in absence of light induce no toxicity. Delivery of PS using internalizing biparatopic nanobody-PS conjugates results in even more pronounced toxicities. Initial in vivo studies point towards a potent therapy, enabling the combination with molecular imaging, which can have a significant impact in the field of targeted PDT. http://dx.doi.org/10.1016/j.pdpdt.2015.07.059 Ru(II) complexes as potent photosensitizers in Photodynamic Therapy

Mn-doped Zinc Sulphide nanocrystals for immunofluorescent labeling of epidermal growth factor receptors on cells and clinical tumor tissues

The field of molecular detection and targeted imaging has evolved considerably with the introduction of fluorescent semiconductor nanocrystals. Manganese-doped zinc sulphide nanocrystals (ZnS:Mn NCs), which are widely used in electroluminescent displays, have been explored for the first time for direct immunofluorescent (IF) labeling of clinical tumor tissues. ZnS:Mn NCs developed through a facile wet chemistry route were capped using amino acid cysteine, conjugated to streptavidin and thereafter coupled to biotinylated epidermal growth factor receptor (EGFR) antibody utilizing the streptavidin–biotin linkage. The overall conjugation yielded stable EGFR antibody conjugated ZnS:Mn NCs (EGFR ZnS:Mn NCs) with a hydrodynamic diameter of 65 ± 15 nm, and having an intense orange–red fluorescence emission at 598 nm. Specific labeling of EGF receptors on EGFR+ve A431 cells in a co-culture with EGFR−ve NIH3T3 cells was demonstrated using these nanoprobes. The primary antibody conjugated fluorescent NCs could also clearly delineate EGFR over-expressing cells on clinical tumor tissues processed by formalin fixation as well as cryopreservation with a specificity of 86% and accuracy of 88%, in comparison to immunohistochemistry. Tumor tissues labeled with EGFR ZnS:Mn NCs showed good fluorescence emission when imaged after storage even at 15 months. Thus, ZnS nanobioconjugates with dopant-dependent and stable fluorescence emission show promise as an efficient, target-specific fluorophore that would enable long term IF labeling of any antigen of interest on clinical tissues.

Nanobody–photosensitizer conjugates for targeted photodynamic therapy

Photodynamic therapy (PDT) induces cell death through light activation of a photosensitizer (PS). Targeted delivery of PS via monoclonal antibodies has improved tumor selectivity. However, these conjugates have long half-lives, leading to relatively long photosensitivity in patients. In an attempt to target PS specifically to tumors and to accelerate PS clearance, we have developed new conjugates consisting of nanobodies (NB) targeting the epidermal growth factor receptor (EGFR) and a traceable PS (IRDye700DX). These fluorescent conjugates allow the distinction of cell lines with different expression levels of EGFR. Results show that these conjugates specifically induce cell death of EGFR overexpressing cells in low nanomolar concentrations, while PS alone or the NB–PS conjugates in the absence of light induce no toxicity. Delivery of PS using internalizing biparatopic NB–PS conjugates results in even more pronounced phototoxicities. Altogether, EGFR-targeted NB–PS conjugates are specific and potent, enabling the combination of molecular imaging with cancer therapy. From the Clinical EditorThis study investigates the role of EGFR targeting nanobodies to deliver traceable photosensitizers to cancer molecules for therapeutic exploitation and concomitant imaging. Altogether, EGFR-targeted NB–PS conjugates combine molecular imaging with cancer therapy, the method is specific and potent, paving the way to clinical application of this technology.

A Novel Method for the Assessment of Targeted PEI-Based Nanoparticle Binding Based on a Static Surface Plasmon Resonance System

The delivery of nucleic acids is a major hurdle in gene therapy or therapeutic gene knockdown, and the development of intelligent and safe nanoparticles as carrier systems is thus under intense investigation. The introduction of ligands for their targeted delivery is of major interest. Here, we describe a novel approach for the analysis of the binding properties of antibody-functionalized nanoparticles, using surface plasmon resonance (SPR) in a static cuvette system. By chemical coupling of the Epidermal Growth Factor Receptor (EGFR)-specific antibody cetuximab to poly(ethylene imine) (PEI) via a PEG-spacer and subsequent DNA or siRNA complexation, we generated targeted nanoplexes with low surface charge. Antibody-mediated uptake into EGFR overexpressing cells was observed. SPR measurements with use of a novel, protein A-based sandwich system for the immobilization of the target receptor in its correct steric orientation allowed the analysis of the specific PEI-PEG-cetuximab binding to EGFR and the determination of binding affinities. Importantly, our cuvette-based SPR assay system was also suitable for the monitoring of ligand-mediated nanoparticle binding, without convection or shear stress. We conclude that our SPR sandwich system allows the precise analysis of the binding of ligand-functionalized nanoparticles in real-time, and we thus establish SPR for the in vitro evaluation of ligand modifications for generating targeted nanoparticles.

EGFR overexpressing cells and tumors are dependent on autophagy for growth and survival

Background and purposeThe epidermal growth factor receptor (EGFR) is overexpressed, amplified or mutated in various human epithelial tumors, and is associated with tumor aggressiveness and therapy resistance. Autophagy activation provides a survival advantage for cells in the tumor microenvironment. In the current study, we assessed the potential of autophagy inhibition (using chloroquine (CQ)) in treatment of EGFR expressing tumors. Material and methodsQuantitative PCR, immunohistochemistry, clonogenic survival, proliferation assays and in vivo tumor growth were used to assess this potential. ResultsWe show that EGFR overexpressing xenografts are sensitive to CQ treatment and are sensitized to irradiation by autophagy inhibition. In HNSSC xenografts, a correlation between EGFR and expression of the autophagy marker LC3b is observed, suggesting a role for autophagy in EGFR expressing tumors. This observation was substantiated in cell lines, showing high EGFR expressing cells to be more sensitive to CQ addition as reflected by decreased proliferation and survival. Surprisingly high EGFR expressing cells display a lower autophagic flux. ConclusionsThe EGFR high expressing cells and tumors investigated in this study are highly dependent on autophagy for growth and survival. Inhibition of autophagy may therefore provide a novel treatment opportunity for EGFR overexpressing tumors.

Scheduling of paclitaxel and gefitinib to inhibit repopulation for optimal treatment of human cancer cells and xenografts that overexpress the epidermal growth factor receptor

In clinical studies, evaluating the combination of chemotherapy and the epidermal growth factor receptor (EGFR) inhibitor gefitinib, treatments were administered concurrently, despite it being counter-intuitive to give a cytostatic agent concurrent with cycle-active chemotherapy. One strategy to enhance efficacy might be to give the agents sequentially, thus allowing selective inhibition of repopulation of cancer cells between doses of chemotherapy. Here, we evaluate the hypothesis that sequential administration might allow inhibition of repopulation by gefitinib, with tumor cells re-entering cycle to allow sensitivity to subsequent chemotherapy. Sequential and concurrent administration of paclitaxel and gefitinib were studied in vitro and in xenografts using EGFR over-expressing, EGFR-mutant, and EGFR wild-type human cancer cell lines. We evaluated cell cycle distribution and repopulation during treatment. The sequential use of gefitinib and paclitaxel to treat EGFR over-expressing A431 cells in vitro decreased repopulation compared to chemotherapy alone, and there was greater cell kill compared to concurrent treatment. In contrast, combined treatment led to greater growth delay than use of gefitinib alone for concurrent but not for sequential treatment of mice bearing A431 xenografts; concurrent treatment had greater effects to reduce functional vasculature in the tumors. Conversely, sequential treatment led to greater growth delay than concurrent treatment of EGFR-mutant HCC-827 xenografts that are sensitive to lower doses of gefitinib. These studies highlight the importance of considering effects on the cell cycle, and on the solid tumor microenvironment, including tumor vasculature, when scheduling cytostatic and cytotoxic agents in combination.

Soluble epidermal growth factor receptor isoforms in non-small cell lung cancer tissue and in blood

Epidermal growth factor receptor (EGFR) is implicated in tumor development and is highly expressed in many human tumors. EGFR overexpression has been observed in both premalignant lesions and in malignant lung tumors, as well as in 40–80% of patients with non-small cell lung cancer (NSCLC). EGFR is a 170-kDa transmembrane glycoprotein with an extracellular ligand-binding domain and a cytoplasmic domain with intrinsic tyrosine kinase activity. Soluble forms of EGFR (sEGFR) containing the extracellular domain have been described both in conditioned media from EGFR overexpressing cells as well as in peripheral blood. However, very little is known regarding the molecular function and the biochemical properties of these circulating EGFR isoforms.This study investigates the expression of sEGFR in lung cancer cultured cells and NSCLC patients with the aim of identifying clinically relevant isoforms specifically produced by tumor cells.Proteomic approaches including OFFGEL electrophoresis and Western blotting analysis were used to assess the sEGFR expression pattern in primary lung tumor samples, normal counterparts and matched plasma.We discover that the isoelectric points of sEGFR isoforms in NSCLC biopsy tissue differ from those of the isoforms present in healthy tissue and detected in the plasma of all subjects.These results demonstrate, for the first time, the existence of sEGFR isoforms specifically produced by NSCLC tumor cells which could represent a new potential biomarker for diagnosis and therapy of lung tumors. However, our observations indicate that more highly sensitive and specific quantitative assays are needed in order to reliably detect the tumor-associated sEGFR isoforms in plasma samples.

Peptide-directed HPMA copolymer-doxorubicin conjugates as targeted therapeutics for colorectal cancer

Synthetic oligopeptides have emerged as a promising class of targeting ligands, providing a variety of choices for the construction of conjugates for desired ligand functionality. To explore the potential of short peptides as ligands for targeted delivery of macromolecular therapeutics for colorectal cancer (CRC), fluorescently labelled HPMA copolymers—bearing either G3-C12 or GE11 for targeting galectin-3 and epidermal growth factor receptor (EGFR), respectively—were synthesised and the mechanisms of their internalisation and subcellular fate in CRC cells were studied. The targetability of the G3-C12 bearing copolymers towards galectin-3 was further compared to that of galactose-containing copolymers. The resulting G3-C12-bearing conjugate actively and selectively targets CRC tumour cells over-expressing galectin-3 and exhibits superior targetability to galectin-3 when compared to the galactose-bearing copolymer. GE11 copolymer conjugate binds specifically and efficiently to EGFR over-expressing cells, thus mediating internalisation to a significantly higher extent relative the copolymer conjugated to a scrambled sequence peptide. We further incorporated doxorubicin (DOX) into GE11 bearing copolymer via an acid-labile hydrazone bond. The GE11-DOX copolymer conjugate demonstrated higher cytotoxicity toward EGFR over-expressing cells relative to the control non-targeted DOX conjugate. Altogether, our results show a proof of principle for the selective delivery of DOX to the target CRC cells.

PIK3CA mutations and EGFR overexpression predict for lithium sensitivity in human breast epithelial cells

A high frequency of somatic mutations has been found in breast cancers within the gene encoding the catalytic p110α subunit of PI3K, PIK3CA. Using isogenic human breast epithelial cells, we have previously demonstrated that oncogenic PIK3CA "hotspot" mutations predict for response to the toxic effects of lithium. However, other somatic genetic alterations occur within this pathway in breast cancers, and it is possible that these changes may also predict for lithium sensitivity. We overexpressed the epidermal growth factor receptor (EGFR) into the non-tumorigenic human breast epithelial cell line MCF-10A, and compared these cells to isogenic cell lines previously created via somatic cell gene targeting to model Pten loss, PIK3CA mutations, and the invariant AKT1 mutation, E17K. EGFR overexpressing clones were capable of cellular proliferation in the absence of EGF and were sensitive to lithium similar to the results previously seen with cells harboring PIK3CA mutations. In contrast, AKT1 E17K cells and PTEN -/- cells displayed resistance or partial sensitivity to lithium, respectively. Western blot analysis demonstrated that lithium sensitivity correlated with significant decreases in both PI3K and MAPK signaling that were observed only in EGFR overexpressing and mutant PIK3CA cell lines. These studies demonstrate that EGFR overexpression and PIK3CA mutations are predictors of response to lithium, whereas Pten loss and AKT1 E17K mutations do not predict for lithium sensitivity. Our findings may have important implications for the use of these genetic lesions in breast cancer patients as predictive markers of response to emerging PI3K pathway inhibitors. See commentary: Treating PIK3CA and EGFR overexpressing breast cancers with lithium citrate

RNA interference for inhibiting expression of epidermal growth factor receptor in breast cancer cells

Objective To investigate whether RNA interference (RNAi) produced by small inter- ference RNA (siRNA) could induce gene silencing in breast cancer cells and to assess the degree of epidermal growth factor (EGF) receptor gene silencing and its effect on functional outcome. Methods Overexpression of EGF receptor gene was determined in breast cancer cells and a variety of other types of cancer cells. Then breast cancer cell lines MDA-MB231, MDA-MB-453S, ZR-75-30, and ZR-75 were transfected with target se- quence-specific dsRNA as well as various controls. Immune fluorescent labeling, flowcytometry and Western blot were used to monitor the reduction in production of the EGF receptor protein. Quantitative reverse- transcriptase PCR was used to detect the silencing of EGF receptor gene levels. Cell count, colony assay, scratch assay, MTT assay, cell cycle analysis, and ELISA were used to assess the functional effects of RNAi. Results EGF receptor gene was overexpressed in breast cancer cells. In cell line MDA-MB231 and MDA-MB-453S, we displayed sequence specific silencing of the EGF receptor with 71.31% of downregulation of EGF receptor protein production and 37.04% of silencing of EGF receptor gene. The reduction in EGF receptor caused growth inhibition of the cells, reducing the total cell numbers by 85.0% and colony number by 63.3%. This also retarded the migration of MDA-MB231 and MDA-MB-453S by more than 80% at 24h and 48h, enhanced the chemosensitivity to cisplatin by four-fold. Cell cycle analysis showed that dsRNA-EGFR induced accumulation of cells in Go-Gi phase by 12.67% with a decrease in the percentage of cells in S-phase by 6.56% relative to control. The results from ELISA showed EGF level was reduced by 27.74% and 11.07% in medium and cell extraction respectively. In cell line ZR-75-30 and ZR-75, we displayed sequence specific silencing of the EGF receptor with 71.78% of downregulation of EGF receptor protein production and 50.00% of silencing of EGF receptor gene. The reduction in EGF receptor caused growth inhibition of the cells, reducing the total cell numbers by 78.3% and colony number by 66.8%. This also retarded the migration of ZR-75-30 and ZR-75 by more than 80% at 24h and 48h, enhanced the chemosensitivity to cisplatin by seven-fold. Cell cycle analysis showed that dsRNA-EGFR induced accumulation of cells in G0-G1 phase by 17.48% with a significant decrease in the percentage of cells in S-phase by 19.20% relative to control. ELISA showed EGF level was reduced by 45.65% and 22.45% in medium and cell extraction, respectively. Conclusions These sequence specific siRNAs showed a blockbuster effect in downregulation of EGF receptor gene expression, inhibition of the cellular proliferation and motility, arrest of the cell cycle, and enhancement of the cellular chemosensitization. The successful application of dsRN A-EGFR to reverse the neoplastic phenotype of EGF receptor overexpressing cells extends the list of available therapeutic modalities in the treatment of human cancer. Key words: Epidermal growth factor receptor; RNA interference; Small interference RNA; Double stranded RNA; Breast cancer

SPECT Imaging with 99mTc-Labeled EGFR-Specific Nanobody for In Vivo Monitoring of EGFR Expression

Overexpression of the epidermal growth factor receptor (EGFR) occurs with high incidence in various carcinomas. The oncogenic expression of the receptor has been exploited for immunoglobulin-based diagnostics and therapeutics. We describe the use of a llama single-domain antibody fragment, termed Nanobody, for the in vivo radioimmunodetection of EGFR overexpressing tumors using single photon emission computed tomography (SPECT) in mice. Fluorescence-activated cell sorting (FACS) analysis was performed to evaluate the specificity and selectivity of 8B6 Nanobody to bind EGFR on EGFR overexpressing cells. The Nanobody was then labeled with (99m)Tc via its C-terminal histidine tail. Uptake in normal organs and tissues was assessed by ex vivo analysis. In vivo tumor targeting of (99m)Tc-8B6 Nanobody was evaluated via pinhole SPECT in mice bearing xenografts of tumor cells with either high (A431) or moderate (DU145) overexpression of EGFR. FACS analysis indicated that the 8B6 Nanobody only recognizes cells overexpressing EGFR. In vivo blood clearance of (99m)Tc-8B6 Nanobody is relatively fast (half-life, 1.5 h) and mainly via the kidneys. At 3 h postinjection, total kidney accumulation is high (46.6+/-0.9%IA) compared to total liver uptake (18.9+/-0.6%IA). Pinhole SPECT imaging of mice bearing A431 xenografts showed higher average tumor uptake (5.2+/-0.5%IA/cm(3)) of (99m)Tc-8B6 Nanobody compared to DU145 xenografts (1.8+/-0.3%IA/cm(3), p<0.001). The EGFR-binding Nanobody investigated in this study shows high specificity and selectivity towards EGFR overexpressing cells. Pinhole SPECT analysis with (99m)Tc-8B6 Nanobody enabled in vivo discrimination between tumors with high and moderate EGFR overexpression. The favorable biodistribution further corroborates the suitability of Nanobodies for in vivo tumor imaging.

Assessment of epidermal growth factor receptor (EGFR, ErbB1) and HER2 (ErbB2) protein expression levels and response to lapatinib (Tykerb, GW572016) in an expanded panel of human normal and tumour cell lines.

Lapatinib (Tykerb, GW572016), a potent inhibitor of the catalytic activities of epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) (ErbB2), inhibits population growth of selected EGFR and HER2 overexpressing cell lines. Previous studies with a small number of cell lines suggest a correlation between overexpression of EGFR and/or HER2 and sensitivity to growth inhibition by lapatinib; however, the precise determinants of lapatinib selectivity for tumour and/or other cells remain unclear. To clarify the determinants of its selectivity in cultured cells, lapatinib-induced cell population growth inhibition and relative EGFR and HER2 protein expression were quantified in 61 different human tumour cell lines from 12 tumour types, two oncogene transformed human cell lines and two normal human cell cultures. Using statistical tools to analyse the data, a model describing the relationship between lapatinib IC(50) (the response variable) and EGFR and HER2 expression and tissue type (explanatory variables) was derived. The results suggest that simultaneous consideration of EGFR and HER2 expression, as well as tissue type yields the best determinant of lapatinib selectivity in cultured cells.

Cytochrome P450 2C9-derived epoxyeicosatrienoic acids induce angiogenesis via cross-talk with the epidermal growth factor receptor (EGFR).

Cytochrome P450 (CYP) epoxygenase products, such as 11,12-epoxyeicosatrienoic acid (EET), stimulate endothelial cell proliferation. We set out to identify the signal transduction cascade linking EET generation to enhanced proliferation and angiogenesis. In human endothelial cells overexpressing CYP 2C9, cell number was increased compared with control cells and was inhibited by the CYP 2C9 inhibitor, sulfaphenazole. CYP 2C9 overexpression was associated with the activation of Akt and an increase in cyclin D1 expression, effects that were abolished by the epidermal growth factor (EGF) receptor inhibitor, AG1478, which also prevented the CYP 2C9-induced increase in cell proliferation. Stimulation of EGF receptor overexpressing cells with 11,12-EET or transfection of these cells with CYP 2C9 enhanced the tyrosine phosphorylation of the EGF receptor. Endothelial tube formation in a fibrin gel was significantly enhanced (6-fold) in CYP 2C9 overexpressing cells and was comparable with the tube formation induced by EGF. In the chick chorioallantoic membrane, 11,12-EET stimulated vessel formation (3.5-fold) and induced vessel convergence, an effect that was abolished by cotreatment with either an EGF receptor-neutralizing antibody or AG1478. These results indicate that CYP 2C9-derived EETs stimulate angiogenesis by a mechanism involving the activation of the EGF receptor.

Epidermal growth factor receptor induced apoptosis: potentiation by inhibition of Ras signaling

Previous studies have shown that certain tumor cell lines which naturally express high levels of the epidermal growth factor receptor (EGFR) undergo apoptosis when exposed to epidermal growth factor. Whether this phenomenon is a direct result of receptor overexpression or some other genetic alteration renders these cells sensitive to apoptosis is yet to be established. We show that experimentally increasing the level of EGFR expression predictably leads to apoptosis in a variety of cell types which requires an active tyrosine kinase but not EGFR autophosphorylation sites. Expression of a dominant negative Ras mutant in EGFR overexpressing cells results in a significant potentiation of EGFR induced apoptosis suggesting that Ras activation is a key survival signal generated by the EGFR. We propose that potentiation of EGFR induced apoptosis by dominant negative Ras results, at least in part, by a block of Akt activation.

Overexpression of epidermal growth factor receptor in human head and neck squamous carcinoma cell lines correlates with matrix metalloproteinase-9 expression andin vitro invasion

Numerous reports have shown an association between overexpression of the epidermal growth factor receptor (EGFR), and poor prognosis in head and neck squamous cell carcinomas (HNSCC), however, the underlying mechanisms are still unclear. In the present study, we set out to determine whether EGFR expression was associated with in vitro invasive capacity in a panel of four established and ten newly derived HNSCC lines. Ten of the cell lines expressed high levels of EGFR as determined by a ligand-binding assay and dot blot analysis, whereas the remaining four showed weak overexpression or normal levels of EGFR. The ability of cells to invade through Matrigel was found to be higher in the EGFR overexpressing cell lines (p < 0. 0001). Expression levels of matrix metalloproteinases (MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, MMP-10, MMP-11, MMP-13, MT1-MMP) and tissue inhibitors of MMP (TIMP-1, TIMP-2) were evaluated by semiquantitative RT-PCR, substrate zymography and western blot. We found a strong positive correlation between EGFR levels and the expression of MMP-9 mRNA (r(2) = 0.95; p < 0.0001), MMP-9 enzyme activity (r(2) = 0.8099; p < 0.0001) and an inverse correlation with TIMP-1 (r(2) = 0.48; p = 0.0059). In six selected HNSCC lines, in vitro invasion was assayed in the presence of an anti-EGFR monoclonal antibody, ICR62. A significant reduction of invasion in four selected EGFR-overexpressing cell lines was found with 30 nM ICR62 (from 50% to 70%; p < 0.001) but there was no effect in two cell lines with normal EGFR levels. Our results show that the in vitro invasive phenotype of HNSCC lines correlates with high EGFR and MMP-9 expression, and it is therefore suggested that the EGFR signaling pathway might play an important role in the invasive behavior of HNSCC via specific upregulation of MMP-9 and downregulation of TIMP-1.

Differential dose-dependent effects of epidermal growth factor on gene expression in A431 cells: evidence for a signal transduction pathway that can bypass Raf-1 activation.

Epidermal growth factor (EGF), which plays an important role in normal and tumoral cell growth regulation, displays an ambivalent dose-dependent effect on the proliferation of epithelial cells overexpressing EGF receptor. However, the underlying molecular mechanisms remain obscure. In this study we have examined the regulation of amphiregulin (AR) gene expression by growth inhibitory (10(-9) M) and stimulatory (10(-12) M) EGF concentrations in A431 cells. The time course of AR messenger RNA (mRNA) accumulation was different with 10(-12) and 10(-9) M EGF; AR induction by 10(-9) M EGF peaked between 1 and 1.5 h, then decreased to the basal level within 2 h. Conversely, the induction by 10(-12) M EGF was slightly delayed, but persisted for 4 h. The involvement of tyrosine phosphorylation in AR induction by EGF was suggested by the ability of the tyrosine phosphatase inhibitor sodium orthovanadate to prolong AR expression induced by 10(-12) or 10(-9) M EGF. In the presence of the protein phosphatase 2A inhibitor, okadaic acid, 10(-9) M EGF induced a persistent accumulation of AR mRNA. On the contrary, okadaic acid abrogated the stimulation of AR mRNA level induced by a low EGF concentration, suggesting that both EGF concentrations activated distinct regulatory mechanisms. The signaling components involved in the differential activities of EGF in A431 cells were then examined. We previously reported a relationship between the ambivalent activity of EGF and the p42-mitogen-activated protein (MAP) kinase activity. Thus, 10(-12) M EGF induced a sustained MAP kinase activation, whereas 10(-9) M EGF led to a sharp, but transitory, activation. The MAP kinases are activated by MAP kinase kinases (MEK1 and MEK2). Whereas no significant effect of 10(-12) M EGF could be detected, 10(-9) M EGF was shown to activate MEK1 and, to a lesser extent, MEK2. Also, both MAP kinase activation and AR induction by 10(-9) M, but not by 10(-12) M, EGF were inhibited by the MEK1 inhibitor PD98059. Moreover, the involvement of c-Raf-1 in the signaling pathway induced by EGF was verified. A concentration of 10(-9) M EGF induced stimulation of c-Raf-1 kinase activity, whereas 10(-12) M EGF not only failed to activate c-Raf-1, but led to a moderate decrease in its kinase activity. These results demonstrate that in EGF receptor-overexpressing cells, EGF may differently affect gene expression and cell proliferation through distinct mechanisms of regulation.